Oligonucleotides, i.e. compounds consisting of from about 4 to about 100 nucleotide units linked together to form a nucleotide chain, are effectively portions of nucleic acids eg. the biologically and genetically important deoxyribonucleic acids (DNA) and ribonucleic acids (RNA). Thus they constitute biologically important products, e.g. as genes. Synthesis of oligonucleotides with exact, predetermined sequences of units in the chain provides materials capable of being spliced into natural nucleic acids in living organisms, e.g. to replace defective natural genetic material or to modify a living cell so as to provide it with the capability to produce useful secretory products on an enhanced scale. For example, insulin-producing genes, synthetic oligonucleotides of correct unit sequence, have been synthesized and introduced into appropriate cells, to enhance insulin production thereby.
It is however necessary to provide reliable, economical processes for producing oligonucleotides, if such production procedures are to assume commercial significance. Synthetic oligonucleotides must be carefully prepared, in step-wise, controlled fashion, to ensure the production of a material with the exactly predetermined unit sequence in its chain. The presence of erroneous chain sequences, omission of a unit from its proper position along the oligonucleotide chain and similar, apparently minor defects, can have potentially disastrous consequences, since the unit sequences in the chain carry the information determining the nature of the secretory products and other behaviour of the cell into which it has been introduced.
One promising method for producing oligonucleotides of predetermined sequence is the polymer support synthesis, described in U.S. patent application Ser. No. 6,149,685, of Ogilvie and Bender, filed May 14, 1980. In this method, a solid polymer such as silica gel is derivatized to put functional side groups thereon, and the polymer is reacted with a first, appropriately protected nucleoside unit, to bond that unit to the solid polymer "support". Then, after suitable deprotection of the linked unit, it is reacted with an appropriate preformed phosphorylated nucleotide compound, which links to the appropriately deprotected location on the polymer-supported unit and hence is added to the nucleotide chain. By repeating the sequence of operations, a nucleotide chain can be built up of substantially any desired length, and with predetermined unit sequences. As a final or close to final step, the oligonucleotide chain is cleaved from the polymer and isolated.
A particular advantage of the above-described process is that it provides the possibility of semi-automatic operation. The polymer-supported product, which is solid, can be kept in a single reaction vessel such as a column throughout the operations, and the various reagents, solvents and wash liquids added thereto from storage vessels of programmed sequence and for pre-set durations. The nucleotide reagents for addition to the growing nucleotide chain are only a total of four in number (for a DNA-type chain or an RNA-type chain, although not the same four in each case). They can be prepared ahead of time, ready for addition to the polymer-supported intermediate product, stored in reagent vessels, and fed to the reaction column as and when required.
In this and similar processes, therefore, the pre-formed polyphorylated nucleotide compounds provide key reagents. They may correspond in general to the formula ##STR1## where R' represents a protecting group for the 5'-hydroxyl and B' represents the base group. The chlorine group is available for reaction with a hydroxyl or another unit, for chain extension purposes. Such compounds are comparatively easy to prepare, but are not very stable.
It would greatly simplify and enhance the economics of the aforementioned and other oligonucleotide preparation processes if phosphorylated nucleotide compounds could become a commodity product, available from central manufacturing sources. However, at present their lack of adequate stability precludes this. They must effectively be prepared as and when required for use in oligonucleotide synthesis, which requires their preparation on site. They are normally too unstable to permit their preparation, storage and transportation prior to use. To have to store and ship the reagents under special conditions to prevent deterioration thereof, or to react the reagents chemically so as to protect their reactive sites and prevent deterioration thereof, is clearly undesirable, as adding additional process steps or apparatus features to the synthesis as a whole.